in organic chemistry · in organic chemistry volume 3-building blocks, catalysis and...

Edited byAndrew B. Hughes

Amino Acids, Peptides and Proteins

in Organic Chemistry

Volume 3 - Building Blocks, Catalysis and

Coupling Chemistry

WILEY-VCH

WILEY-VCH Verlag GmbH & Co. KG aA

Contents

List of Contributors XVII

Part One Amino Acids as Building Blocks 1

1 Amino Acid Biosynthesis 3

Emily J. Parker and AndrewJ. Pratt

1.1 Introduction 3

1.2 Glutamate and Glutamine: Gateways to Amino Acid Biosynthesis 5

1.2.1 Case Study: GOGAT: GATs and Multifunctional Enzymesin Amino Acid Biosynthesis 6

1.3 Other Amino Acids from Ubiquitous Metabolites: PyridoxalPhosphate-Dependent Routes to Aspartate, Alanine, and Glycine 8

1.3.1 Pyridoxal Phosphate: A Critical Cofactor ofAmino Acid Metabolism 8

1.3.2 Case Study Dual Substrate Specificity of Families

ofAminotransferase Enzymes 10

1.3.3 PLP and the Biosynthesis ofAlanine and Glycine 15

1.4 Routes to Functionalized Three-Carbon Amino Acids: Serine,

Cysteine, and Selenocysteine .76

1.4.1 Serine Biosynthesis 16

1.4.2 Cysteine Biosynthesis 18

1.4.3 Case Study Genome Information as a Starting Point for UncoveringNew Biosynthetic Pathways 19

1.4.3.1 Cysteine Biosynthesis in Mycobacterium Tuberculosis 19

1.4.3.2 Cysteine Biosynthesis in Archaea 20

1.4.3.3 RNA-Dependent Biosynthesis of Selenocysteine and Other

Amino Acids 21

1.5 Other Amino Acids from Aspartate and Glutamate: Asparagine

and Side Chain Functional Group Manipulation 22

1.5.1 Asparagine Biosynthesis 23

Amino Acids, Peptides and Proteins in Organic Chemistry.Vol.3 - Building Blocks, Catalysis and Coupling Chemistry. Edited by Andrew B. HughesCopyright © 20U WILEY-VCH Veriag GmbH & Co. KGaA, WeinheimISBN: 978-3-527-32102-5

1.6 Aspartate and Glutamate Families of Amino Acids 25

1.6.1 Overview 25

1.6.2 Aspartate Family Amino Acids: Threonine and Methionine 25

1.6.2.1 Case Study: Evolution of Leaving Group Specificity in Methionine

Biosynthesis 28

1.6.2.2 Threonine, Homocysteine, and PLP 30

1.6.2.3 Threonine Synthase 30

1.6.2.4 Methionine, Cysteine, and Cystathionine 32

1.6.2.5 Methionine Synthase 33

1.6.3 Glutamate Family Amino Acids: Proline and Arginine 33

1.7 Biosynthesis ofAliphatic Amino Acids with Modified Carbon Skeletons:

Branched-Chain Amino Acids, Lysine, and Pyrrolysine 37

1.7.1 Overview 37

1.7.2 Valine and Isoleucine 37

1.7.3 Homologation of a-Keto Acids, and the Biosynthesis of Leucine

and a-Aminoadipic Acid 41

1.7.4 Biosynthesis of Lysine: A Special Case 44

1.7.4.1 Diaminopimelate Pathway to Lysine 44

1.7.4.2 a-Aminoadipic Acid Pathways to Lysine 45

1.7.4.3 Pyrrolysine 47

1.8 Biosynthesis of the Aromatic Amino Acids 49

1.8.1 Shikimate Pathway 49

1.8.2 Case Study Alternative Synthesis of Dehydroquinate in Archaea 53

1.8.3 Biosynthesis of Tryptophan, Phenylalanine, and Tyrosine from

Chorismate 58

1.8.3.1 Tryptophan Biosynthesis 58

1.8.3.2 Phenylalanine and Tyrosine Biosynthesis 59

1.8.4 Histidine Biosynthesis 61

1.9 Conclusions 64

References 65

2 Heterocycles from Amino Acids 83

M. Isabel Calaza and Carlos Cativiela

2.1 Introduction 83

2.2 Heterocycles Generated by Intramolecular Cyclizations 83

2.2.1 a-Lactones and a-Lactams 83

2.2.2 Indolines 84

2.2.3 Aziridinecarboxylic Acids and Oxetanones 86

2.2.4 |3-Lactams and Pyroglutamic Acid Derivatives 87

2.2.5 Amino Lactams and Amino Anhydrides 88

2.2.6 Azacycloalkanecarboxylic Acids 89

2.3 Heterocycles Generated by Intermolecular Cyclizations 89

2.3.1 Metal Complexes 89

2.3.2 a-Amino Acid N-Carboxyanhydrides and Hydantoins 90

2.3.3 Oxazolidinones and Imidazolidinones 91

2.3.4 Oxazolones 93

2.3.5 Oxazinones and Morpholinones, Pyrazinonesand Diketopiperazines 94

2.3.6 Tetrahydroisoquinolines and B-Carbolines 96

2.3.7 Oxazo/Thiazolidinones, Oxazo/Thiazolidines,and Oxazo/Thiazolines 97

2.3.8 Sulfamidates 101

2.3.9 Tetrahydropyrimidinones 102

2.4 Heterocycles Generated by Cycloadditions 202

2.5 Conclusions 104

2.6 Experimental Procedures 104

2.6.1 Synthesis of l-tert-Butyl-3-phenylaziridinone (5) 104

2.6.2 Synthesis of Dimethyl (2S,3ai?,8aS)-l,2,313a,8,8a-

Hexahydropyrrolo[2,3-b]indole-l,2-dicarboxylate (Precursor of 10) 105

2.6.3 Synthesis of Benzyl (R)-l-Tritylaziridine-2-carboxylate (15) 105

2.6.4 Synthesis of (S)-N-tert-Butoxycarbonyl-3-aminooxetan-2-one (18) 106

2.6.5 Synthesis of (S)-l-(tert-Butyldimethylsilyl)-4-oxoazetidine-2-carboxylic Acid (24) 106

2.6.6 Synthesis of9H-Fluoren-9-ylmethyl (£)-Hexahydro-2-oxo-lH-azepin-3-yl Carbamate (31) 106

2.6.7 Synthesis of Ethyl (S)-N-(tert-Butoxycarbonyl)-a-

(tert-butoxymethyl)proline Ester (36) 107

2.6.8 Synthesis of Proline N-Carboxyanhydride (49) 107

2.6.9 Synthesis of (2S,4S)-2-Ferrocenyl-3-pivaloyl4-methyl-l,3-oxazolidin-5-one (54b) 107

2.6.10 Synthesis of (6S)-6-Isopropyl-5-phenyl-3,6-dihydro-2H-l,4-oxazin-2-one (71) 108

2.6.11 Synthesis of (3S,6J?)-6-Isopropyl-3-methyl-5-phenyl-l,2,3,6-tetrahydro-2-pyrazinone (78) 108

2.6.12 Synthesis of (3S)-3,6-Dihydro-2,5-dimethoxy-3-

isopropylpyrazine (85) 109

2.6.13 Synthesis of (3S)-l,2,3,4-Tetrahydroisoquinoline-3-carboxylic Acid (87) 110

2.6.14 Synthesis of Methyl (S)-N-tert-Butoxycarbonyl-2,2-dimethyloxazolidine-4-carboxylate (109) 110

2.6.15 Synthesis of (2S,6S)-2-tert-Butyl-l-carbobenzoxy-4-oxopyrimidin-6-

carboxylic Acid (126) 111

References 111

3 Radical-Mediated Synthesis ofot-Amino Acids and Peptides 115

Jan Deska

3.1 Introduction 135

3.2 Free Radical Reactions 115

3.2.1 Hydrogen Atom Transfer Reactions 116

3.2.2 Functional Group Transformations 121

3.3 Radical Addition to Imine Derivatives 124

3.3.1 Glyoxylate Imines as Radical Acceptors 125

3.3.2 Oximes and Hydrazones as Radical Acceptors 126

3.3.3 Nitrones as Radical Acceptors 129

3.3.4 Isocyanates as Radical Acceptors 130

3.4 Radical Conjugate Addition 130

3.5 Conclusions 135

3.6 Experimental Protocols 135

3.6.1 Preparation of ((li?,2S,5R)-5-methyl-2-(l-methyl-l-

phenylethyl)cyclohexyl 2-[(tert-butoxycarbonyl)amino]-4-methyl-

pent-4-enoate) (7) 135

3.6.2 Synthesis of (2S)-3-{(lJR,2S)-2-[(N-bis-Boc)amino]-l-cyclopropyl}-2-benzyloxycarbonylamino-propionic Acid Methyl Ester (26) 136

3.6.3 Synthesis of (3ai?,6S,7aS)-hexahydro-8,8-dimethyl-l-[(2i?)-3,3-dimethyl-l-oxo-2-(2,2-diphenylhydrazino)butyl]-3H-3a,6-methano-2,l-benzisothiazole 2,2-dioxide (42) 136

3.6.4 Synthesis of N-(2,6-diphenyl-methylpiperidine-2-carboxamide (59) 137

3.6.5 Synthesis of Methyl 2-(2-naphthylcarbonylamino)pentanoate(80) 136

References 138

4 Synthesis of ^-Lactams (Cephalosporins) by Bioconversion 143

Jose" Luis Barredo, Maria Rodriguez- Sdiz, Josd Luis Adrio,and Arnold L. Dema'm


4.2 Biosynthetic Pathways of Cephalosporins and Penicillins 146

4.3 Production of 7-ACA by A. chrysogenum 147

4.4 Production of 7-ADCA by A. chrysogenum 149

4.5 Production of Penicillin G by A. chrysogenum 151

4.6 Production of Cephalosporins by P. chrysogenum 152

4.7 Conversion of Penicillin G and other Penicillins to DAOG

by Streptomyces clavuligems 153

4.7.1 Expandase Proteins and Genes 153

4.7.2 Bioconversion of Penicillin G to DAOG 155

4.7.3 Broadening the Substrate Specificity of Expandase 155

4.7.3.1 Resting Cells 255

4.7.3.2 Cell-Free Extracts 156

4.7.4 Inactivation of Expandase during the Ring-Expansion Reaction 157

4.7.5 Further Improvements in the Bioconversion of Penicillin

G to DAOG 158

4.7.5.1 Stimulatory Effect of Growth in Ethanol 158

4.7.5.2 Use of Immobilized Cells 159

4.7.5.3 Elimination ofAgitation and Addition of Water-Immiscible

Solvents 259

4.7.5.4 Addition of Catalase 260

4.7.5.5 Recombinant S. clavuligerus Expandases 260

4.8 Conclusions 162

References 163

5 Structure and Reactivity of ^-Lactams 169

Michael I. Page5.1 Introduction 169

5.2 Structure 270

5.3 Reactivity 174

5.4 Hydrolysis 276

5.4.1 Base Hydrolysis 276

5.4.2 Acid Hydrolysis 278

5.4.3 Spontaneous Hydrolysis 280

5.4.4 Buffer-Catalyzed Hydrolysis 280

5.4.5 Metal Ion-Catalyzed Hydrolysis 280

5.4.6 Micelle-Catalyzed Hydrolysis of Penicillins 182

5.4.7 Cycloheptaamylose-Catalyzed Hydrolysis 284

5.4.8 Enzyme-Catalyzed Hydrolysis 285

5.4.8.1 Serine ^-Lactamases 285

5.4.8.2 Metallo P-Lactamases 287

5.5 Aminolysis 292

5.6 Epimerization 295

References 295

Part Two Amino Acid Coupling Chemistry 202

6 Solution-Phase Peptide Synthesis 203

Yuko Tsuda and Yoshio Okada

6.1 Principle of Peptide Synthesis 203

6.2 Protection Procedures 205

6.2.1 Amino Group Protection 205

6.2.1.1 Z Group 205

6.2.1.2 Substituted Z and other Urethane-Type Protecting Groups 207

6.2.1.3 Boc Group 207

6.2.1.4 Tri Group 208

6.2.1.5 Fmoc Group 209

6.2.1.6 Other Representative Protecting Groups 222

6.2.2 Carboxyl Group Protection 222

6.2.2.1 Methyl Ester (-OMe) and Ethyl Ester (-OEt) 213

6.2.2.2 Benzyl Ester (-OBzl) 223

6.2.2.3 iBu Ester (-OtBu) 223

6.2.2.4 Phenacyl Ester (-OPac) 224

6.2.2.5 Hydrazides 224

6.2.3 Side-Chain Protection 225

6.2.3.1 e-Amino Function of Lys (8-Amino Function of Orn) 235

6.2.3.2 fJ-Mercapto Function of Cys 216

6.2.3.3 fi-and Y-Carboxyl Functions of Asp and Glu 217

6.2.3.4 Protecting Groups for the y-Carboxyl Function of Glu 219

6.2.3.5 8-Guanidino Function of Arg 219

6.2.3.6 Phenolic Hydroxy Function ofTyr 221

6.2.3.7 Aliphatic Hydroxyl Function of Ser and Thr 222

6.2.3.8 Imidazole Nitrogen of His 222

6.2.3.9 Indole Nitrogen of Trp 223

6.3 Chain Elongation Procedures 223

6.3.1 Methods of Activation in Stepwise Elongation 223

6.3.1.1 Carbodiimides 223

6.3.1.2 Mixed Anhydride Method 224

6.3.1.3 Active Esters 225

6.3.1.4 Phosphonium and Uronium Reagents 227

6.3.2 Methods of Activation in Segment Condensation 229

6.3.2.1 Azide Procedure 229

6.3.2.2 Carbodiimides in the Presence of Additives 230

6.3.2.3 Native Chemical Ligation 231

6.4 Final Deprotection Methods 232

6.4.1 Final Deprotection by Catalytic Hydrogenolysis 233

6.4.2 Final Deprotection by Sodium in Liquid Ammonia 233

6.4.3 Final Deprotection by TFA 233

6.4.4 Final Deprotection by HF 233

6.4.5 Final Deprotection by HSAB Procedure 234

References 234

7 Solid-Phase Peptide Synthesis: Historical Aspects 253

Garland R. Marshall


7.2 Selection of Compatible Synthetic Components 253

7.3 Racemization and Stepwise Peptide Assembly 257

7.4 Optimization of Synthetic Components 258

7.5 Foreshadowing of the Nobel Prize 258

7.6 Automation of SPPS 260

7.7 Impact of New Protecting Groups and Resin Linkages 261

7.8 Solid-Phase Organic Chemistry 262

7.Si Early Applications of SPPS to Small Proteins 263

7.10 Side-Reactions and Sequence-Dependent Problems 264

7.11 Rapid Expansion of Usage Leading to the Nobel Prize 265

7.12 From the Nobel Prize Forward to Combinatorial

Chemistry 267

7.13 Protein Synthesis and Peptide Ligation 268

7.14 Conclusions 269

References 270

8 Linkers for Solid-Phase Peptide Synthesis 273

Miroslav Soural,Jan Hlavdc, and Viktor Krchhdk


8.1.1 Immobilization Strategies 275

8.1.2 Overview of Linker Types 276

8.1.3 Selection of a Linker 277

8.2 Immobilization via Carboxyl Group 279

8.2.1 Esters 280

8.2.1.1 Hydroxy Linkers for Preparation ofResin-Bound Esters 281

8.2.1.2 Electrophilic Linkers for Preparation of Resin-Bound Esters 282

8.2.1.3 Cleavage from the Resin 282

8.2.2 Amides 288

8.2.3 Hydrazides 291

8.2.4 Oximes 291

8.2.5 Thioesters 292

8.3 Immobilization via Amino Group 294

8.4 Backbone Immobilization 296

8.4.1 Benzaldehyde-Based Linkers 298

8.4.2 Indole Aldehyde Linkers 299

8.4.3 Naphthalene Aldehyde Linkers (NALs) 299

8.4.4 Thiophene Aldehyde Linkers (T-BALs) 300

8.4.5 Safety-Catch Aldehyde Linkers 300

8.4.6 Photolabile Aldehyde Linker (PhoB) 300

8.5 Immobilization via Amino Acid Side-Chain 300

8.5.1 Carboxyl Group 301

8.5.2 Amino and Other Nitrogen-Containing Groups 302

8.5.2.1 Lys 302

8.5.2.2 His 302

8.5.2.3 Arg 303

8.5.3 Hydroxy Group 303

8.5.4 Sulfanyl Group 304

8.5.5 Aromatic Ring 305

8.6 Conclusions 306

References 306

9 Orthogonal Protecting Croups and Side-Reactions in Fmoc/iBuSolid-Phase Peptide Synthesis 313

Stefano Carganico and Anna Maria Papini9.1 Orthogonal Protecting Groups in Fmoc/tBu Solid-Phase Peptide

Synthesis 313

9.1.1 Arg 313

9.1.2 AsnandGln 325

9.1.3 AspandGlu 326

9.1.4 Cys 318

9.1.5 His 323

9.1.6 Lys 324

9.1.7 Met 327

9.1.8 SerandThr 327

9.1.9 Trp 328

9.1.10 Tyr 329

9.1.11 Conclusions 330

9.2 Side-Reactions in Fmoc/iBu Solid-Phase Peptide Synthesis 330

9.2.1 Imidazole Ring-Mediated Racemization of Chiral a-Carbon 332

9.2.2 Hydroxyl-Mediated O -+ N Acyl Transfer 332

9.2.3 Met Oxidation to Methionyl Sulfoxide 334

9.2.4 Dehydration ofAsn and Gin Amide Side-Chain 334

9.2.5 Aspartimide Formation 336

9.2.6 Formation of Diketopiperazines 337

9.2.7 Side-Reactions Affecting Protected Cys 338

9.2.8 Deletion Peptides, Truncated Sequences, and MultipleAdditions 338

9.2.9 Uronium/Guanidinium Salts-Induced Guanidino

Capping 340

9.2.10 Arg Cyclization and Arg Conversion into Orn 341

9.2.11 Conclusions 342

References 343

10 Fmoc Methodology: Cleavage from the Resin

and Final Deprotection 349

Fernando Albericio, Judit Tulla-Puche, and Steven A. Kates


10.2 "Low" TFA-Labile Resins 351

10.2.1 Cleavage 351

10.2.2 Choice of Resin for the Preparation of Peptide Acids 352

10.2.2.1 CTC Resin 353

10.2.2.2 SASRIN Resin 355

10.2.2.3 Bromide Resin 356

10.2.3 Final Deprotection 356

10.3 "High" TFA-Labile Resins 356

10.3.1 Cleavage 357

10.3.2 Final Deprotection of Protected Peptides in Solution 359

10.3.3 Side-Reactions 360

10.3.3.1 Linker/Resin 360

10.3.3.2 Trp and Tyr Modification 362

10.3.3.3 Sulfur-Containing Residues: Cys and Met 362

10.3.3.4 Ser andThi, N—> O Migration 363

10.3.3.5 Asp and Asn 363

10.3.3.6 Arg 364

10.3.3.7 N-Alkylamino Acids 365

10.3.3.8 Work-Up 366

10.4 Final Remarks 366

References 366

H Strategy in Solid-Phase Peptide Synthesis 371

Kleomenis Barlos and Knut Adermann

11.1 Synthetic Strategies Utilizing Solid-Phase Peptide SynthesisMethods 371

11.2 Solid Support: Resins and Linkers 373

11.3 Developing the Synthetic Strategy: Selection ofthe ProtectingGroup Scheme 374

11.4 Resin Loading 376

11.5 SBS Peptide Chain Elongation: Coupling and Activation 377

11.6 Piperazine Formation 378

11.7 Solid-Phase Synthesis of Protected Peptide Segments 379

11.8 Fragment Condensation Approach: Convergent and Hybrid

Syntheses 379

11.9 Cleavage from the Resin and Global Peptide Deprotection 382

11.10 Disulfide Bond-Containing Peptides 384

11.11 Native Chemical Ligation (NCL) 386

11.12 SPPS of Peptides Modified at their C-Terminus 388

11.13 Side-Chain-Modified Peptides 390

11.14 Cyclic Peptides 392

11.15 Large-Scale Solid-Phase Synthesis 394


References 396

12 Peptide-Coupling Reagents 407

Ayman El- Faham and Fernando Albericio


12.2 Carbodiimides 409

12.2.1 General Procedure for Coupling Using Carbodiimide and HOXt;

Solution Phase 423

12.2.1.1 General Procedure for Solid-Phase Coupling via Carbodiimide

Activation 424

12.2.2 Loading ofWang Resin Using Carbodiimide 415

12.3 Phosphonium Salts 416

12.3.1 Preparation of Phosphonium Salts 418

12.3.2 General Method for the Synthesis of Phosphonium Salts 420

12.4 Aminium/Uronium Salts 420

12.4.1 Stability of Onium Salts 425

12.4.2 General Procedure for the Preparation of Chloroformamidinium

Salts 426

12.4.3 Synthesis of Aminium/Uronium Salts 427

12.4.4 General Procedure for Coupling Using Onium Salts

(Phosphonium and Uronium) in Solution Phase 427

12.4.5 General Procedure for Coupling Reaction in Solid-Phase UsingOnium Salts (Phosphonium and Uronium) 427

12.4.6 General Procedures for Coupling Reaction in Solid-Phase UsingOnium Salts (Phosphonium and Uronium) Boc-, Fmoc-Amino

Acids via Phosphonium and Uronium Salts 427

12.5 Fluoroformamidinium Coupling Reagents 429

12.5.1 General Method for the Synthesis of Fluoroformamidinium

Salts 431

12.5.2 Solution- and Solid-Phase Couplings via TFFH 432

12.5.3 General Method for Solid-Phase Coupling via TFFH 432

12.6 Organophosphorus Reagents 432

12.6.1 General Method for Synthesis ofthe DiphenylphosphorylDerivatives 435

12.7 Triazine Coupling Reagents 435

12.7.1 Formation of the Peptide Bond Using DMTMM (128) 437

12.8 Mukaiyama's Reagent 437


References 439

13 Chemoselective Peptide Ligation: A Privileged Tool for

Protein Synthesis 445

Christian P. R. Hackenberger, Jeffrey W. Bode, and Dirk Schwarzer


13.2 Chemoselective Peptide Ligations Following a Capture/Rearrangement Strategy 449

13.2.1 Basic Concepts and Early Experiments 449

13.2.2 NCL 452

13.2.3 Protein Semisynthesis with NCL 454

13.2.4 Protein Semisynthesis with Expressed Protein Ligation 456

13.2.5 Protein Trans-Splicing 457

13.3 Chemical Transformations for Cys-Free Ligations in Peptidesand Proteins 460

13.3.1 Chemical Modification of NCL Products 460

13.3.1.1 Desulfurization 463

13.3.1.2 Alkylation and Thioalkylation Protocols 464

13.3.2 Auxiliary Methods 466

13.3.2.1 (Oxy-)Ethanethiol Auxiliary 467

13.3.2.2 Photoremovable Na-l-Aryl-2-Mercaptoethyl Auxiliary 468

13.3.2.3 4,5,6-Trimethoxy-2-Mercaptobenzylamine Auxiliary 468

13.3.2.4 Sugar-Assisted Glycopeptide Ligations 469

13.4 Other Chemoselective Capture Strategies 472

13.4.1 Traceless Staudinger Ligation 472

13.4.1.1 Imine Ligations with Subsequent Pseudo-Pro Formation 473

13.5 Peptide Ligations by Chemoselective Amide-Bond-FormingReactions 474

13.5.1 Thio Acid/Azide Amidation 475

13.5.2 Thio Acid/N-Arylsulfonamide Ligations 475

13.5.3 Chemoselective Decarboxylative Amide Ligation 477

13.6 Strategies for the Ligation of Multiple Fragments 479

13.6.1 Synthetic Erythropoietin 480

13.6.2 Convergent Strategies for Multiple Fragment Ligations 480

13.6.2.1 Ubiquitylated Histone Proteins 484

References 486

14 Automation of Peptide Synthesis 495

Carlo Di Bello, Andrea Bagno, and Monica Dettin


14.2 SPPS: From Mechanization to Automation 497

14.3 Deprotection Step: Monitoring and Control 500

14.4 Coupling Step: Monitoring and Control 505

14.5 Integrated Deprotection and Coupling Control 509

References 514

15 Peptide Purification by Reversed-Phase Chromatography 519

Ulrike Kusebauch, Joshua McBee, Julie Bletz, Richard J. Simpson,and Robert L. Moritz

15.1 RP-HPLC ofPeptides 519

15.2 Peptide properties 520

15.3 Chromatographic Principles 520

15.3.1 Choice of Mobile Phase 520

15.3.1.1 Mobile-Phase Aqueous Buffer pH 520

15.3.1.2 Organic Solvent 522

15.3.2 Stationary Phase 523

15.3.2.1 Surface Bonding 523

15.3.2.2 Pore Diameter 523

15.3.2.3 Particle Size 524

15.3.2.4 Ultra-High-Pressure Liquid Chromatography 525

15.3.2.5 Synthetic Polymer Packings 525

15.3.2.6 Monolithic Stationary Phase 525

15.3.2.7 Packed Bed (Column) Length 526

15.3.2.8 Gradient Effect 527

15.3.2.9 Temperature 527

15.4 Prediction of Peptide Retention Times 528

15.5 Advantages of Reduced Scale 531

15.6 Two-Dimensional Chromatographic Methods 532

15.7 Peptide Analysis in Complex Biological Matrices 533

15.8 Standard Methods for Peptide Separations for Analysis by

Hyphenated Techniques 534

15.9 Emerging Methods for Peptide Separations for Analysis by

Hyphenated Techniques 534

15.10 Practical use of RP-HPLC for Purifying Peptides (Analyticaland Preparative Scale) 539

15.10.1 Simple Protocol for Successful RP-HPLC 540

15.10.1.1 Buffer Preparation 540

15.10.1.2 HPLC Chromatographic System 542

15.10.1.3 Test Sample 542

References 544

16 Difficult Peptides 549

M. Teresa Machini Miranda, Cleber W. Una, and Cesar Remuzgo16.1 Importance ofPeptide Synthesis 549

16.2 Methods for Peptide Synthesis 550

16.3 Chemical Peptide Synthesis 551

16.4 "Difficult Peptide Sequences" 554

16.5 Means to Overcome Peptide Aggregation in SPPS 556

16.5.1 In Situ Neutralization 556

16.5.2 Solvents for Peptide Chain Assembly 557

16.5.3 Type and Substitution Degree of Resins for Peptide Chain

Assembly 557

16.5.4 Use of Chaotropic Salts During Peptide Chain Assembly 558

16.5.5 Use of Amide Backbone Protection 558

16.5.6 The Use ofPseudo-Prolines 560

16.5.7 O-Acyl Isopeptide Approach 561

16.5.8 Use of Elevated Temperatures 562

16.6 Monitoring the Synthesis of a "Difficult Peptide" 562


References 564

Index 571

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